Detection of antibodies to gangliosides using solid-phase reactants coated with carbonyl groups

ABSTRACT

Methods of detecting antibodies to one or more ganglioside(s) of interest in a sample are disclosed which comprise using a solid-phase reactant having carbonyl groups attached thereon, and the ganglioside(s) of interest linked to the solid-phase reactant by an amide bond between an amino group of the ganglioside of interest and a carbonyl group attached to the solid-phase reactant. The methods of detecting antibodies to ganglioside(s) of interest can be used in methods of diagnosing neuropathies in an individual.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/565,837, filed May 5, 2000. The entire teachings of the aboveapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Antibodies to gangliosides have been implicated in many differentautoimmune neuropathies. For example, serum IgM antibodies to GMganglioside have been found in a patient with lower motor neuronsyndrome (Adams, D. et al., J. Neurol. Neurosurg. Psychiatry 56:982-987(1993)), and are common in multifocal motor neuropathy (Komberg, A. J.and Pestronk, A., Ann. Neurol. 37:S43-S50 (1995); Komberg, A. J. andPestronk, A., Muscle Nerve 17:100-104 (1994); Cetacea, S. A. et al.,Neurology 40:1067-1072 (1990); Adams, D. et al., Neuroimmunology32:223-230 (1991); Taylor, B. V. et al., Neurology 46:951-955 (1996)).Autoantibodies against GM- and GM-gangliosides have been found inconjunction with chronic idiopathic demyelinating polyneuropathy (CID)in conjunction with systemic lupus erythematosus (SLE) (Sindem, E. etal., Acta. Neural. Scand. 83:399-402 (1991)). Anti-GQIB gangliosideantibodies were identified in patients with Fisher's syndrome (Yuri, N.et al., Neurology 43:414-417 (1993)). Sera from patients withGillian-Barre syndrome has been found to have antibodies to variousgangliosides, including antibodies to GM1B ganglioside (see, e.g.,Cathouse, K. et al., J. Neurol. Sci. 156:99-101 (1998)).

Enzyme-linked immunosorbent assays (ELISA) have been used foridentification of antibodies to gangliosides (see, for example,identification of GM ganglioside in Pestronk, A. et al. Ann. Neurol.27:316-326 (1990)); U.S. Pat. No. 5,443,952; Aenaeus-Kanaf, A. B. etal., Neurochem. Inv. 20(3):353-357 (1992)). However, high backgroundvalues frequently interfere with accurate assessment of the amount ofanti-ganglioside antibodies (Ravindranath, M H et al., J ImmunologicalMethods 169:257-272 (1994)). Reliable measurement of anti-gangliosideantibodies is critical for correct diagnosis of neuropathies,particularly motor neuropathies.

SUMMARY OF THE INVENTION

The present invention pertains to methods of determining, in a testsample, the amount of antibodies directed against a specific nervoussystem antigen or antigens, using a modified solid-phase reactant. Themethod utilizes a solid-phase reactant, such as a microtiter plate, thatis modified with carbonyl groups attached to its surface. One or moregangliosides of interest (e.g., asialo GM ganglioside, GM1 ganglioside,GM2 ganglioside, GM3 ganglioside, GD1a ganglioside, GD1B ganglioside,GD2 ganglioside, GD3 ganglioside, GQ1b ganglioside, and/or GT1bganglioside) are linked to the modified solidphase reactant by an amidebond between an amino group of the ganglioside and a carbonyl groupattached to the solid-phase reactant. One or more control antigens, suchas other glycolipids, glycoproteins or carbohydrates, can also beattached on the surface of the modified solid-phase reactant. Themodified solid-phase reactant having ganglioside(s) of interest linkedthereon is contacted with a test sample, such as a test sample of abodily fluid (e.g., blood, serum, cerebrospinal fluid, or urine) from anindividual, under conditions such that any antibody to theganglioside(s) of interest that may be present in the test sample canbind to the ganglioside(s) of interest linked to the modifiedsolid-phase reactant. The amount of antibodies in the test sample to theganglioside(s) of interest is then determined using standard methods,such as enzyme-linked immunosorbent assay (ELISA) or another appropriatesolid-phase assay. If a control antigen is attached on the modifiedsolid-phase reactant, the level of antibodies in the test sample to thecontrol antigen, can also be determined using the same methods. Specificreactivity of antibodies to the ganglioside of interest is determined bythe amount of antibody binding to the ganglioside of interest that isabove the amount of antibody binding to the control antigen. The methodscan be used for diagnosing a neuropathy in an individual. The amount ofantibody to a ganglioside of interest in a test sample from theindividual is determined using the methods. An amount of antibody to aganglioside of interest that is greater, by an amount that isstatistically significant, than the amount of antibody to theganglioside of interest in a control sample, is indicative of thepresence of the neuropathy. Alternatively, an amount of antibody to aganglioside of interest that is equal to or greater than an establishedreference amount is indicative of the presence of the neuropathy.

The invention also pertains to test kits, containing modifiedsolid-phase reactants, for use in the methods of the invention.

The high sensitivity and specificity of the methods can clarify thedifferential diagnosis of neuropathies and reduce the need fortime-consuming and expensive electrophysiological evaluation.Furthermore, a modified solid-phase reactant having carbonyl groupsattached to its surface allows the use of a smaller amount ofganglioside than the amount which would otherwise be necessary toperform similar assays with a solid-phase reactant not having thismodification. In addition, a modified solid-phase reactant havingcarbonyl groups attached to its surface can be coated with a gangliosideof interest without a need for toxic solvents; the coating is notaffected by humidity, and yields consistent and reproducible assayresults.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic depiction of a comparison of signal to backgroundnoise of several types of solid phase reactants. Black, signal; grey,background.

FIG. 2 is a graphic depiction of a comparison of sensitivity oftraditional solid phase reactants to modified solid phase reactants indetecting titers of antibodies to ganglioside. Grey, traditional solidphase reactant; black, modified solid phase reactant.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein relates to methods of determining theamount of antibodies to one or more ganglioside(s) of interest in asample. The invention further pertains to methods of diagnosingneuropathies (e.g., multifocal motor neuropathy) in an individual bydetermining the amount of antibodies to one or more ganglioside(s) ofinterest in a sample from the individual. Applicant has discovered thatsignificantly increased sensitivity for antibodies to certaingangliosides can be achieved by conducting an enzymelinked immunosorbentassay (ELISA) using microtiter plates that are modified with carbonylgroups, allowing amide linkage of gangliosides to the plates.

As a result of this discovery, highly sensitive and specific methods ofdetermining the presence or absence, and the amount, of antibody to oneor more ganglioside(s) of interest in a sample are now available. In themethods, a modified solid-phase reactant is used. The term, “solid-phasereactant”, as used herein, refers to a solid medium, such as amicrotiter plate, a membrane (e.g., nitrocellulose), a bead, a dipstick,a thin-layer chromatographic plate, or other solid medium. In apreferred embodiment, the solid-phase reactant is a microtiter platethat can be used in a solid-phase immunoassay, such as an enzyme-linkedimmunosorbent assay. The solid-phase reactant is modified such thatcarbonyl groups are grafted into the reactant. As a result of thepresence of the carbonyl groups, certain antigens, such as glycolipids,glycoproteins, carbohydrates, or other antigens containing amino groups,can be linked on the surface of the solid-phase reactant by an amidebond between the carbonyl group on the solid-phase reactant and theamino group of the antigen. A representative solid-phase reactant thatallows linkage of antigens on its surface in this manner is the Co-starDNA-BIND covalent plate (Co-star, Corning, N.Y.). A solid-phase reactantthat has carbonyl groups attached thereon and therefore has the abilityto allow amide bond linkage of antigens onto its surface is referred toherein as a “modified solid phase reactant”.

One or more ganglioside(s) of interest is linked to the modifiedsolid-phase reactant. A ganglioside of interest that is “linked” or“attached” to the modified solid-phase reactant is a ganglioside thathas formed an amide bond between an amino group of the ganglioside andthe carbonyl group attached to the modified solid-phase reactant.Representative gangliosides include asialo GM ganglioside, GM1ganglioside, GM2 ganglioside, GM3 ganglioside, GDIa ganglioside, GDIBganglioside, GD2 ganglioside, GD3 ganglioside, GQ1b ganglioside, andGTIb ganglioside. One type of ganglioside can be used; alternatively,more than one type of ganglioside can be linked to the covalent-linkagesolid phase reactant. As used herein, a solid-phase reactant having “atleast one” ganglioside of interest linked thereon may have only one typeof ganglioside of interest thereon, or may also have more than one typeof ganglioside of interest thereon. In a preferred embodiment, asialo GMganglioside; GM1 ganglioside; and GD1b ganglioside are linked to themodified solidphase reactant. A representative method of linking usesthe ganglioside of interest reconstituted in methanol and diluted intophosphate buffered saline. EDAC(1-ethyl-3-(3diethylaminopropyl)carbodiimide) can also be included, ifdesired; for example, in one embodiment, if the ganglioside of interestis GDIB ganglioside, EDAC is included in the solution containing theganglioside; the solution containing the ganglioside is then allowed tocoat the modified solid-phase reactant. Because the modified solid-phasereactant contains carbonyl groups, amino groups present on thegangliosides form amide bonds with the carbonyl groups when exposed tothe carbonyl groups on the modified solid-phase reactant. A modifiedsolid-phase reactant on which one or more ganglioside(s) is attached isreferred to herein as a “ganglioside modified solid-phase reactant”.

A control antigen, such as another ganglioside, or a glycolipid,glycoprotein or carbohydrate, can also be attached to the modifiedsolid-phase reactant. More than one control antigen can be used. Acontrol antigen can be identified, for example, by evaluation of anumber of samples from individuals having known disease states.“Specific binding” is indicated by statistically demonstrated binding ofantibody in the sample to the antigen of interest, relative to theclinical status (disease state) of the samples (e.g., binding, in astatistically significant number of samples from individuals having aparticular disease state, of antibody to the particular antigen). A lackof binding to a particular antigen by a sample from an individual havinga known clinical status is generally accepted as being indicative of anon-reactive (control) antigen.

The control antigen(s) can be attached to the modified solid-phasereactant using methods similar to those used to coat the ganglioside(s)of interest onto the modified solidphase reactant. The control antigenis usually attached to the modified solid-phase reactant at a differentlocation than the ganglioside(s) of interest. For example, if thesolid-phase reactant is a microtiter plate, a ganglioside of interestcan be attached to certain wells of the plate, and the control antigencan be attached to other wells of the plate. In another example, if morethan one ganglioside of interest is attached to the plate, the controlantigen is attached to certain wells of the plate; a first gangliosideof interest is attached to other wells of the plate; a secondganglioside of interest is attached to different wells of the plate fromeither the control antigen or the first ganglioside of interest, etc.Alternatively, the control antigen can be attached to a separatesolid-phase reactant, the separate solid-phase reactant being the sametype of solid-phase reactant as that onto which the ganglioside(s) ofinterest is coated. It is intended that the term, “ganglioside modifiedsolid-phase reactant”, refers to those modified solid-phase reactantshaving one or more ganglioside(s) of interest attached thereon, as wellas to those modified covalent-linkage solid-phase reactants having oneor more ganglioside(s) of interest attached thereon as well as one ormore control antigens attached at a different location thereon. Theterm, “control antigen solid-phase reactant” is used to refer to asolid-phase reactant having solely control antigen(s) attached thereto.

The ganglioside modified solid-phase reactant (and control antigensolid-phase reactant, if used) is used in an assay to determine theamount of antibody to one or more gan.glioside(s) of interest in a testsample. The test sample to be assayed for the amount of antibody to aganglioside of interest can be a sample of bodily fluid or tissue froman individual For example, the test sample can comprise blood, serum,cerebrospinal fluid, urine, nasal secretion, saliva, or any other bodilyfluid or tissue. Alternatively, the test sample can comprise antibodies,and particularly 1gM, IgG and/or IgA antibodies, that have been isolatedfrom a sample of bodily fluid or tissue from the individual. In apreferred embodiment, the test sample is a serum sample from anindividual suspected of having a neuropathy.

To determine the amount of anti-ganglioside antibody in a test sample,the ganglioside modified solid-phase reactant is contacted with the testsample. A ganglioside modified solid-phase reactant that has beencontacted with a test sample is referred to herein as a “contactedganglioside modified solid-phase reactant,” The contacted gangliosidemodified solid-phase reactant is maintained under appropriate conditionsto allow binding of any antibody to the ganglioside(s) of interest thatmay be present in the test sample, to the ganglioside(s) of interestthat is attached to the solid-phase reactant. The term, “antibody to aganglioside of interest” refers to an antibody or antibodies thatpreferentially binds to the ganglioside of interest. For example, anantibody to a ganglioside of interest preferentially binds to theganglioside of interest in an amount that is greater than to controlantigens (e.g., glycolipids, glycoproteins or carbohydrates), and/or inan amount that is greater than to other gangliosides, by an amount thatis statistically significant. The antibody to the ganglioside ofinterest may bind to a ganglioside that is in a lipid environment (e.g.,GM1 ganglioside in a lipid mixture of GM1 ganglioside,galactocerebroside, and cholesterol (GGC)), and/or may bind to aganglioside that is isolated (e.g., not in a lipid environment).

The amount of antibody to the ganglioside(s) of interest in the testsample, if any, that has bound to the ganglioside(s) of interest on themodified solid-phase reactant is determined. The amount is determinedseparately for each ganglioside of interest. It is expected that anantibody will specifically interact with a ganglioside of interest; thatis, an antibody will interact preferentially with one ganglioside ofinterest, and not to another ganglioside of interest.

The amount of antibody can be determined by a variety of methods usingstandard techniques, including enzyme-linked immunosorbent assay(ELISA), solid phase radioimmunoassay, or other solid phase immunoassays(see Ausubel, F. M. et al., eds., Current Protocols in MolecularBiology, John Wiley & Sons, 1996, especially units 11.2 (ELISA) and11.16 (Determination of Specific Antibody Titer); the entire teachingsof this reference are incorporated herein by reference). In a typicalsolid-phase immunoassay, the amount of antibody bound to the gangliosideof interest attached to the modified solid-phase reactant is determinedusing a developing reagent, such as a detection antibody that binds tothe antibody to the ganglioside of interest. The detection antibody canbe linked or conjugated to another molecule, such as an enzyme orfluorophore, to facilitate detection. Alternatively, the detectionantibody is iodinated. If more than one ganglioside of interest is used,different detection antibodies can be used to detect each antibody toeach ganglioside of interest. For example, a detection antibody thatbinds to an antibody to one ganglioside of interest can be conjugated toa first fluorophore, and a detection antibody that binds to an antibodyto a second ganglioside of interest can be conjugated to a secondfluorophore that is distinguishable from the first fluorophore.Alternatively, if the same detection reagent is used, the differentgangliosides of interest can be attached to the modified solid-phasereactant at a different, identifiable location, such that the presenceof a detection reagent at a particular location corresponds to thepresence of antibodies to the ganglioside of interest attached to themodified solid-phase reactant at that location.

In a preferred embodiment, an ELISA assay is performed, using as adeveloping reagent a detection antibody that is linked to an enzyme,such as horseradish peroxidase. The contacted ganglioside modified solidphase-reactant is incubated with the developing reagent, forming adeveloped, contacted solid-phase reactant. Subsequently, a substrate ofthe enzyme is added to the developed, contacted solid-phase reactant,and the amount of activity of the enzyme on its substrate (e.g., theamount of hydrolysis of the substrate) is measured by an appropriatemeans, such as by measuring optical density.

Titers of antibodies to the ganglioside(s) of interest can be calculatedfrom the amount of detector antibody bound to the antibodies to theganglioside of interest, using standard conversion algorithms. Forexample, if the developing reagent comprises horseradish peroxidase,titers of antibody can be calculated as set forth in Pestronk et al.(Ann. Neurol. 2.7:316-326 (1990)).

If a control antigen is attached to the modified solid-phase reactant,titers of antibody binding to the control antigen are subtracted fromthe titers of antibody binding to the ganglioside of interest. Thedifference between the titer of antibody binding to the ganglioside ofinterest and the titer of antibody binding to the control antigen(s) isindicative of the specific (selective) binding of the antibody to theganglioside of interest. If the control antigen is attached to aseparate modified solid-phase reactant, the control antigen modifiedsolid-phase reactant is contacted with the test sample in the samemanner as the ganglioside modified solid-phase reactant and maintainedunder the same conditions. The amount of antibody to the control antigenis determined by the same method as is used to determine the amount ofantibody to the ganglioside of interest.

Neuropathies, particularly immune-mediated neuropathies, can bediagnosed using these methods of determining the amount of antibody to aganglioside of interest. To diagnose these neuropathies, the test sampleis a sample from an individual to be tested for the presence of aneuropathy. The amount of antibody to a ganglioside(s) of interest inthe test sample is compared with the amount of comparable antibody tothe ganglioside(s) of interest in at least one comparable control sample(i.e., a sample of the same type(s) of antibody (1gM, IgG, and/or IgA)from an individual who is not afflicted by a neuropathy). The controlsample can be a sample from any individual who is not afflicted with aneuropathy; it is not necessary that the control sample be from anindividual who is free of disease. For example, the control sample canbe a sample from an individual who has amyotrophic lateral sclerosis, orsystemic immune disorders. A “comparable” normal sample is a sample ofthe same type of body fluid or tissue as the test sample; alternatively,if the test sample is IgM antibodies isolated from a sample of fluid ortissue, the comparable normal or control sample is a sample of 1gMantibodies isolated from the same type of bodily fluid or tissue. Morethan one control sample can be used. The presence of an amount ofspecific (selective) ganglioside antibody binding in the test samplethat is greater, by an amount that is statistically significant, thanthe amount of specific (selective) ganglioside antibody binding in acomparable control sample, correlates with a diagnosis of theneuropathy.

Alternatively, the amount of antibody to a ganglioside(s) of interest inthe test sample can be compared with a “reference amount”. A referenceamount, as used herein, is an amount (e.g., a titer) of antibody to aganglioside of interest which has been previously determined tocorrelate with a particular disease state. For example, a referenceamount can be determined by assessing the amount off antibody toganglioside(s) of interest in a set of samples from individuals havingknown diseases (e.g., neuropathies), as well as comparable controlsamples as described above, and determining what amount of antibodycorrelates with disease. An amount of antibody to a ganglioside(s) ofinterest in the test sample that is equal to, or greater than, thereference amount, correlates with a diagnosis of the neuropathy.

The present invention also includes kits to be used in methods of theinvention. Kits can include the following components: (1) a modifiedsolid-phase reactant having carbonyl groups, and also having one or moreganglioside(s) of interest attached thereto by amide 2 0 bonds; and (2)labeled detector antibody that binds to the antibody to theganglioside(s) of interest. The detector antibody can be specific forthe type of antibody (e.g., IgM, IgG or IgA). Detector antibody cancomprise an antibody bound to a detectable agent, such as an enzyme,radioactive molecule, or fluorescent agent. If the detector antibody isbound to an enzyme that reacts with an added substrate to yield acolored product, such as horseradish peroxidase, the kit can alsoinclude the substrate.

The invention is now further illustrated by the followingExemplification.

Exemplification:

Comparison of Antibody Titers Using a Variety of Enzyme LinkedImmunosorbent Assay (ELISA) Plates

A comparison of background noise and antibody titers was made, usingtraditional (Falcon polystyrene) plates, plates modified with carbonylgroups allowing amide linkage of gangliosides to the plates (Co-starDNA-BIND plates), and plates modified with secondary amino groups on itssurface (Nunc CovaLink plates, Nunc, Roskilde, Denmark).

Linking of Antigen to Covalent Plates

Antigen-coating solutions were prepared. For GD1b ganglioside, 1% EDACin PBS (0.01 g EDAC per ml PBS) was used; for GM1 ganglioside and asialoganglioside, PBS was used. Antigen stocks were prepared byreconstituting lyophilized antigen vials with methanol to aconcentration of 1 mg/ml. An aliquot of the 1 mg/ml GD1b stock wasdiluted into 1% EDAC to make GDIb antigen coating solution at thedesired concentration. Aliquots of the 1 mg/ml GM1 ganglioside andasialo ganglioside stocks were diluted in PBS to make the GM1ganglioside and asialo ganglioside coating solutions at the desiredconcentrations.

Each plate required 5.5 ml of coating antigen solutions. Plates wereremoved from foil pouches just prior to use, avoiding direct light thatcould damage the plates. Antigen coating solution (100 ml/well) wasadded to rows A, B, 'B, and F; rows C, D, G and H were left un-coated asthe antigen blank wells. Coated plates were incubated in the dark at 4°C. overnight. The coating solution was then aspirated, and the plateswashed 3 times with 1% BSA. After the third wash, all plate wells werefilled with 1% BSA, and the plates were blocked for at least 16 hours at4° C. in the dark.

Sample Addition

A robot robotic automatic pipeting system was used to add dilutedsamples and controls to the plates. The plates were then incubated at 4°C. overnight in the dark.

Detection Antibody Probe and Colorimetric Reaction

Detection antibody (peroxidase conjugated goat anti-human JgG, 1gM andIgA cocktail) was diluted in 1% BSA to the desired dilution, which wasdetermined for each lot of antibody prior to use. Eleven ml of dilutedetection antibody were used for each plate. Assay plates were aspiratedand washed 6 times with 1% BSA, and then blotted. Dilute detectionantibody solution (100 ml/well) was added to all assay plates, and theplates were incubated in the dark at room temperature for 2 hours.Substrate solution was then prepared: 100 mg of OPD and 12 ml of 30%hydrogen peroxide were added to 100 ml of 0.1 M citric acid (pH 4.5) toform substrate solution (11 ml per assay plate). All assay plates werethen aspirated and washed 6 times with 1% BSA, and then blotted.Substrate solution (100 ml/well) was added to all assay plates, whichwere then covered to avoid light exposure. Pleases were incubated atroom temperature, and read approximately 20 minutes after addition ofsubstrate at 405 mn until both positive controls reached the establishedminimum OD validated for the positive control in use.

Results

A comparison of the background noise of a variety of ELISA~plates wasperformed. A positive clinical control was used to represent the signal,and the background was a BSA buffer blank (absence of sample). Resultsare shown in Table 1 and FIG. 1. TABLE 1 Comparison of Background Noisewith Different ELISA Plates Plate Signal Background Traditional 0.810.05 Co-star DNA-Bind 0.82 0.05 Nunc Covalink 0.82 0.35

FIG. 1 demonstrates the results of the comparison of the signal tobackground noise of the traditional plate (Falcon polystyrene plate);the plate having carbonyl groups attached thereon (the Co-star DNA-BINDplate); and the plate having secondary amino groups on its surface (NUNCCoating plate, Nunc; Roskilde, Denmark). It can be seen that the platehaving carbonyl groups exhibited low background noise compared to theother plates.

Sensitivity of the current methods was also assessed. Six samplespreviously run in a clinically validated GMI ganglioside assay and foundto have low positive anti-GM1 ganglioside antibody were used. Resultsare shown in Table 2 and FIG. 2. TABLE 2 Comparison of Antibody TitersObtained with Different Plates Traditional Plate (Falcon) Covalent Plate(C-Star Patient Number Titer DNA BIND) Titer 1 1600 6400 2 1600 6400 31600 6400 4 3200 12800 5 1600 6400 6 3200 25600

FIG. 2 demonstrates the sensitivity of the current methods. The platehaving carbonyl groups attached thereon yields a consistently highertiter for the same samples. The elevated signal obtained by performingan ELISA using the plate having carbonyl groups attached thereondiminishes “grey areas” (titers that are close to the reference amount,and are therefore considered “borderline”), and thereby decreases thenumber of questionable positive samples. Furthermore, the amount ofganglioside used on the plate having carbonyl groups attached thereonwas half the amount on the traditional Falcon polystyrene plate.

Those skilled in the art will be able to recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1. A method of diagnosing a neuropathy in an individual, comprisingdetermining the amount of antibody to a ganglioside of interest in atest sample from the individual by conducting an assay using a modifiedsolid-phase reactant having carbonyl groups attached thereon and aganglioside of interest linked to the modified solid-phase reactant byan amide bond between an amino group of the ganglioside of interest anda carbonyl group attached to the modified solid-phase reactant, whereinan amount of antibody to a ganglioside of interest that is greater, byan amount that is statistically significant, than the amount in acontrol sample, is indicative of the presence of the neuropathy.
 2. Themethod of claim 1, comprising conducting an enzyme-linked immunosorbentassay to determine the amount of antibody to the ganglioside ofinterest.
 3. The method of claim 1, wherein the solid-phase reactant isa microtiter plate.
 4. The method of claim 1, wherein the test sample isselected from the group consisting of: blood, serum, cerebrospinal fluidand urine.
 5. A method of diagnosing a neuropathy in an individual,comprising determining the amount of antibody to a ganglioside ofinterest in a test sample from the individual by conducting an assayusing a modified solid-phase reactant having carbonyl groups attachedthereon and a ganglioside of interest linked to the modified solid-phasereactant by an amide bond between an amino group of the ganglioside ofinterest and a carbonyl group attached to the modified solid-phasereactant, wherein an amount of antibody to a ganglioside of interestthat is equal to or greater than a reference amount is indicative of thepresence of the neuropathy.
 6. The method of claim 5, comprisingconducting an enzyme-linked immunosorbent assay to determine the amountof antibody to the ganglioside of interest.
 7. The method of claim 5,wherein the solid-phase reactant is a microtiter plate.
 8. The method ofclaim 5, wherein the test sample is selected from the group consistingof: blood, serum, cerebrospinal fluid and urine.
 9. A kit for detectingthe amount of antibody to a ganglioside of interest, comprising amodified solid-phase reactant having carbonyl groups attached thereonand a ganglioside of interest linked to the modified solid-phasereactant by an amide bond between an amino group of the ganglioside ofinterest and a carbonyl group attached to the modified solid-phasereactant.
 10. The kit of claim 9, wherein the solid-phase reactant is amicrotiter plate.
 11. The kit of claim 9, wherein the ganglioside ofinterest is selected from the group consisting of: asialo GMganglioside, GM1 ganglioside, GM2 ganglioside, GM3 ganglioside, GD1aganglioside, GD1B ganglioside, GD2 ganglioside, GD3 ganglioside, GQ1bganglioside, and GT1b ganglioside.